RNAs play key roles in maintenance and expression of genetic information and provide potential targets and reagents for therapeutic intervention in pathological states associated with infectious diseases and hereditary disorders. Assembly of precise RNA structures and RNA-ligand complexes is essential to virtually all RNA-mediated processes. Our goal is to understand how RNAs fold into functional structures in living cells. Direct analysis of RNA folding steps in biological processes is challenging because numerous components interact in complex pathways and many steps intervene between assembly of an RNA structure and execution of its biological function. Catalytic RNAs provide useful model systems for probing folding mechanisms because catalytic activity reports directly and quantitatively on assembly of functional RNA structures. The foundation of our program is a system we developed using RNA catalysis to monitor intracellular assembly of RNA structures that integrates molecular biology and genetics with kinetics and thermodynamics. So far, this unique approach has enabled us to show that some kinetic and equilibrium parameters of intracellular RNA folding reactions agree remarkably well with parameters measured for the same reactions in vitro, provided that in vitro reactions approximate intracellular ionic conditions. On the other hand, we found that competition between alternative RNA secondary structures produces dramatically different outcomes in vitro and in vivo. The discrepancy between in vitro and in vivo RNA folding behavior highlights the importance of investigating RNA folding directly and quantitatively in a biological context. We propose to elaborate upon this approach by incorporating metabolomics and fluorescence methodologies and apply it to study new areas of intracellular RNA folding. These areas include the interplay between kinetics and thermodynamics during mRNA remodeling after transit through the ribosome, the influence of RNA-binding proteins and small ligands on RNA folding during transcription, and the mechanisms through which regulatory RNAs integrate information from multiple chemical signals to regulate gene expression. The proposed studies will generate fundamental insights into folding of the RNA structures that are central to normal growth and development and the assembly of RNA complexes with intracellular ligands that mediate gene regulation. The results of this work will also provide a framework for developing technical and therapeutic applications that involve RNAs as targets and reagents.